Malignant cells selectively express on their surface molecules that have functional importance in cell adhesion, invasion and metastasis. Some of these tumour-associated structures are the result of a blockage in the glycosylation pathway. In particular, the incomplete elongation of O-glycan saccharide chains leads to the expression of shorter carbohydrate structures such as Tn, sialyl-Tn or TF antigens (Hollingsworth and Swanson 2004). The Tn antigen, defined as a GalNAc unit α-linked to a serine or threonine residue (α-GalNAc-O-Ser/Thr), is one of the most specific human tumour-associated structures. Tn is detected in about 90% of human carcinomas (Springer 1984) and its expression is correlated to carcinoma aggressiveness (Springer 1997). Moreover, under appropriate conditions, Tn is capable of inducing a strong immune response in mice and non human primates, the resulting antibodies being capable of recognizing human cancer cells (Lo-Man et al., 2001, Lo-Man et al. 2004).
This O-linked epitope is usually expressed on mucins as their carbohydrate core structure (Hollingsworth and Swanson 2004). Mucins are high molecular weight O-glycosylated proteins (50-80% of their mass is due to O-linked carbohydrate chains) that participate in protection, lubrication and acid resistance of the epithelial surface (Gendler and Spicer 1995). To date, different mucins have been identified and numbered in chronological order of their description (MUC1-MUC20) (Chen et al., 2004, Filshie et al., 1998, Gum et al. 2002, Higuchi et al. 2004, Moniaux et al., 2001, Pallesen et al., 2002, Williams et al., 2001, Yin and Lloyd 2001). Although they do not show homology of sequence, all mucins present a large region composed of variable number of tandem repeats (VNTR). These regions, usually called tandem repeats, are characterized by a high content in serine, threonine (which constitute the potential O-glycosylation sites) and proline residues.
Each organ or tissue exhibits a unique pattern of MUC gene expression (Gendler and Spicer 1995). This mucin expression profile can be modified under pathological conditions and especially during malignant transformation. Upregulation, downregulation, and de novo expression of mucin proteins have been reported in cancer epithelial cells and are thought to influence cell adhesion (Hilkens et al. 1992) and to contribute to tumour invasiveness (Segal-Eiras and Croce 1997). Moreover, these tumour-associated mucins show antigenic differences from normal mucins and are highly immunogenic and as such, they may be used as potential targets for immunotherapy (Agrawal et al. 1998, Apostolopoulos et al. 1996). In particular, MUC1 is undergoing several clinical trials as anti-cancer vaccine (Finn et al. 1995, Gilewski et al., 2000).
MUC6 was first isolated from a human stomach library (Toribara et al. 1993) and it is expressed at high levels in the normal stomach and gall bladder with weaker expression in the terminal ileum, right colon and in the endocervix (De Bolos et al., 1995, Ho et al., 1995, Reis et al. 2000, Toribara et al. 1993). MUC6 has a tandem repeat unit of 169 amino acids (507 bp each) (Toribara et al. 1993) and Southern blot analyses of the shortest MUC6 alleles indicate that they contain at least 15 repeat units (Vinall et al. 1998). Although the whole MUC6 gene was localized and identified, a full length cDNA has not been completely sequenced yet (Rousseau et al. 2004). In addition to its normal expression in gastric tissues, MUC6 has been detected in Barret adenocarcinoma and metasplasia (Guillem et al. 2000), in intestinal adenoma and carcinoma (Guillem et al. 2000), in pulmonary carcinoma (Hamamoto et al. 2005, Nishiumi et al., 2003), in colorectal polyps (Bartman et al. 1999) and in breast carcinoma (De Bolos et al. 1995, Pereira et al., 2001), while it is not expressed in the respective normal tissues. In some cases, MUC6 expression has been reported to be correlated to degrees of histopathology related to malignant potential (Bartman et al. 1999, Hamamoto et al. 2005, Nishiumi et al. 2003). We have recently shown that MUC6 is aberrantly glycosylated in MCF7 breast cancer cells since it contains the Tn antigen (Freire et al. 2005). Several studies have shown that the carbohydrate structures on mucins (including the core Tn antigen) may be essential for the definition of the tumour-associated structures (Grinstead et al., 2003, von Mensdorff-Pouilly et al., 2005). Therefore, Tn-MUC6 glycoconjugates represent attractive targets to be used in cancer immunotherapy. A specific anti-Tn antibody response should target cancer cells through the Tn antigen, which is expressed on their surface. Furthermore, the activation of mucin-specific cytotoxic T lymphocytes should be favoured through the up-take of soluble MUC6-Tn immune complexes by Fc receptors on dendritic cells (Amigorena and Bonnerot 1999).
Prior art techniques however suffer from the drawback of not enabling an easy production of mucin-Tn glycoconjugates.
Prior art mucin-Tn glyconjugates are:                naturally-occurring glyconjugates, or        synthetic glycopeptides.        
Naturally-occurring mucin-Tn glyconjugates are obtained by isolation from a biological source (Podolsky 1985; Robbe et al. 2004). Such glycoconjugates can be obtained only in very low quantities. Their apomucin backbone is a complete apomucin protein, which bear a great number of different carbohydrate residues. The naturally-occurring mucin-Tn glyconjugates not only contain Tn, sTn and TF antigens, but also a great number of other carbohydrate residues, the nature of which varies depending on the type, state, and status of the cell from which they originate.
The preparation of naturally-occurring antigenic glycoconjugates further relies on multi-step tedious and/or time-consuming purifications.
Other prior art mucin-Tn glycoconjugates are synthetic mucin-Tn glycopeptides. Their apomucin backbone is limited to a few amino acids.
For example, Kagan et al. 2005 discloses KHL conjugates of MUC1 or MUC2 glycopeptides. The peptide backbone of these KHL conjugates is a MUC1 or MUC2 32aa peptide, and the enzyme used to glycosylate these peptides is T2 and/or T4 N-acetylgalactosaminyltransferase(s).
Such prior art glycopeptides, when used alone, are not very efficient in inducing an immunogenic response: they require to be conjugated to a protein carrier, such as KLH, to exert their antigenic properties, if any. As a consequence, the mucin-derived glycopeptides used so far as immunogens are in fact KLH conjugates.
Other synthetic mucin glycopeptides have been described by the present inventors, in Freire et al. 2005 (Cancer Res. 65(17): 7880-7887).
Freire et al. 2005 describes the production of a MUC6-Tn glycopeptide (GTTPPPTTLK; SEQ ID NO:14), and of MUC1-Tn, MUC2-Tn, MUC5B-Tn glycopeptides. The apomucin backbone of these mucin-Tn glycoconjugates is a 9-12aa peptide (10aa for MUC6-Tn; 9aa or 11aa for MUC1-Tn; 12aa for MUC2-Tn; 11 for MUC5B-Tn; see page 7881 of Freire et al. 2005).
These mucin-Tn glycopeptides are produced:                either by the quite expensive process of glycopeptide synthesis using a protected glycosylated building block [Fmoc-Thr(α-GalNAc(OAc)3)-OH] at the appropriate place in the peptide sequence (see the paragraph entitled “synthetic (glyco)peptides”, in the “Materials and Methods” section in page 7881),        or by enzymatic transfer of GalNAc into the apomucin peptide, wherein MCF-7 microsome extracts are used as a source of ppGalNAc-T activity, and wherein glycosylation is monitored by reverse-phase HPLC (see the paragraph entitled “Enzymatic transfer of GalNAc or Gal into MUC6 or MUC6-Tn, respectively”, in the “Materials and Methods” section in page 7881; see also FIG. 1 on page 7882).        
Both processes are however limited to the glycosylation of 9-12aa peptides, and do not attain semi-preparative amounts of production (mg to g).
Freire et al. 2005 does further not disclose any immunisation-related result for these MUC6-Tn, MUC1-Tn, MUC2-Tn, MUC5B-Tn glycopeptides, whether linked to a protein carrier such as KLH, or not.
In order to further develop anti-tumour vaccines based on the Tn antigen, the present inventors provide an in vitro enzymatic method for the preparation of Tn-based mucin glycoconjugates, and describe new mucin glycopolypeptides and new immunogenic compositions, which overcome the drawbacks of prior art techniques, and which can induce a highly efficient immunogenic response.
The present inventors developed an enzymatic approach, which enables the production of mucin glycoconjugates with a high Tn density in at least semi-preparative scale amounts.
Contrary to prior art synthetic glycopeptides, the mucin glycoconjugates of the invention are immunogenic, even when used in the absence of any carrier protein.
The immunogenic mucin glycoconjugates of the invention differ from the naturally-occurring glyconjugates in that their carbohydrate component does not have a heterogeneous and variable composition. The carbohydrate component of the immunogenic mucin glycoconjugates of the invention has a precise composition: each of the carbohydrate moieties that are directly O-linked to a Ser or Thr residue of the apomucin backbone is a GalNAc moiety.
To the best of the inventors' knowledge, it is the first work reporting the induction of human tumour cell-specific antibodies after immunization with a mucin derived polypeptide carrying the Tn antigen, without a protein carrier.
As a very advantageous feature, the mucin glycoconjugates produced in accordance with the present invention induce an immunogenic effect that is specific immunogenic effect: upon in vivo administration, the mucin glycoconjugates of the invention are capable of inducing antibodies, and advantageously IgG antibodies, which are capable of recognizing human tumour cells through a Tn-dependent mechanism.